A comparative study of periplast structure inCryptomonas cryophila andC. ovata (Cryptophyceae)

Summary Freeze-fracture followed by deep-etch was used with transmission electron microscopy to characterize and compare the periplasts of two cryptomonads,Cryptomonas ovata andC. cryophila. The periplast ofC. ovata consists of a dense surface mat of granular/fibrillar material overlying a series of polygonal plates attached to the undersurface of the plasma membrane (PM) at their upturned edges. Fracture faces of the PM reveal a highly stable substructure with distinct patterns of intra-membrane particles (IMPs) associated with the underlying plates; a role for the PM in plate development is indicated. The surface periplast component ofC. cryophila exhibits a cover of morphologically complex, overlapping heptagonal scales (termed rosette scales) in addition to elongate fibrils. The arrangement of IMPs within the PM is predominantly random and the inner periplast component consists of a sheet with regular pores where ejectisomes are located. The sheet does not appear closely associated with the PM. The combination of features exhibited by the periplast ofC. cryophila warrants its inclusion as a new type within theCryptophyceae.     

Arabinogalactan-rich glycoproteins are localized on the cell surface and in intravacuolar multivesicular bodies

We investigated the subcellular distribution of antigenic sites immunoreactive to the monoclonal antibody 16.4B4 (PM Norman, VPM Wingate, MS Fitter, CJ Lamb [1986] Planta 167: 452-459) in tobacco (Nicotiana tabacum) leaf cells. This antibody is directed against a glycan epitope in a family of plasma membrane arabinogalactan proteins of 135 to 180 kilodaltons, elaborated from a polypeptide of relative molecular mass 50 kilodaltons (PM Norman, P Kjellbom, DJ Bradley, MG Hahn, CJ Lamb [1990] Planta 181: 365-373). We demonstrated by immunogold electron microscopy that the epitope reactive with monoclonal antibody 16.4B4 is localized on the cell surface in the leaf parenchyma cell periplast. The 16.4B4 antigen is also localized in multivesicular invaginations of the plasma membrane also known as plasmalemmasomes, implying a biochemical and, hence, functional interrelationship between these structures. Monoclonal antibody 16.4B4 also labels intracellular multivesicular bodies that appear to represent internalized plasmalemmasomes. Antibody reactivity was also observed in partially degraded multivesicular bodies sequestered within the central vacuole. We propose that the subcellular distribution of the epitope reactive with monoclonal antibody 16.4B4 defines a plasmalemmasome (or multivesicular body-mediated) pathway for the internalization of the periplasmic matrix for vacuolar mediated disposal. The multivesicular bodies appear to be equivalent to the well-characterized endosomes and multivesicular bodies of animal cells involved in the internalization and lysosome-mediated degradation of extracellular materials.     

Structure and development of the cryptomonad periplast: A review

Summary The structure and development of the complex periplast, or cell covering, of cryptomonads is reviewed. The periplast consists of the plasma membrane (PM) plus an associated surface periplast component (SPC) and cytoplasmic or inner periplast component (IPC). The structure of the SPC and IPC, and their association with the PM, varies considerably between genera. This review, which concentrates on cryptomonads with an IPC of discrete plates, discusses relationships between periplast components and examines the development of this unique cell covering. Formation and growth of inner plates occurs throughout the cell cycle from specialized regions termed anamorphic zones. Crystalline surface plates, which comprise the SPC in many cryptomonad species, appear to form by self-assembly of disorganized subunits. InKomma caudata the subunits are composed of a high molecular weight glycoprotein that is produced within the endomembrane system and deposited onto the cell surface within anamorphic zones. The self-assembly of subunits into highly ordered surface plates appears closely associated with developmental changes in the underlying IPC and PM.     

Periplast structure of the cryptomonad flagellateHemiselmis brunnescens

Summary The periplast ofHemiselmis brunnescens Butcher is a complex cell covering comprised of the plasma membrane (PM) sandwiched between a surface periplast component (SPC) and an inner periplast component (IPC). The SPC is revealed by deep-etching, and consists of hexagonal plates composed of tripartite subunits that appear to self-assemble into a crystalline layer with a hexagonal symmetry. Small scales (termed fibrillar scales) accumulate on the crystalline plates during cell growth, eventually forming a carpet that itself may appear crystalline when fully formed. Heptagonal rosette scales are occasionally observed on the surface as well. The position of the crystalline plates is precisely mirrored by both the E and P fracture faces of the PM. The plate proper is underlain by membrane with a high concentration of intramembrane particles (IMPs) while the bands of membrane underlying the plate borders lack IMPs. Access of subunits and fibrillar scales to the cell surface following initial plate formation appears to be at the plate boundaries. This study suggests that cryptomonad flagellates may provide model systems for studying the self-assembly of cell surface components, and for relating membrane structure to function, as evidence suggests a major role for the PM in mediating periplast assembly and development.     

Structural and biochemical bases of photorespiration in C<Subscript>4</Subscript> plants: quantification of organelles and glycine decarboxylase

In C₄ plants, photorespiration is decreased relative to C₃ plants. However, it remains unclear how much photorespiratory capacity C₄ leaf tissues actually have. We thoroughly investigated the quantitative distribution of photorespiratory organelles and the immunogold localization of the P protein of glycine decarboxylase (GDC) in mesophyll (M) and bundle sheath (BS) cells of various C₄ grass species. Specific differences occurred in the proportions of mitochondria and peroxisomes in the BS cells (relative to the M cells) in photosynthetic tissues surrounding a vein: lower in the NADP-malic enzyme (NADP-ME) species having poorly formed grana in the BS chloroplasts, and higher in the NAD-malic enzyme (NAD-ME) and phosphoenolpyruvate carboxykinase (PCK) species having well developed grana. In all C₄ species, GDC was localized mainly in the BS mitochondria. When the total amounts of GDC in the BS mitochondria per unit leaf width were estimated from the immunogold labeling density and the quantity of mitochondria, the BSs of NADP-ME species contained less GDC than those of NAD-ME or PCK species. This trend was also verified by immunoblot analysis of leaf soluble protein. There was a high positive correlation between the degree of granal development (granal index) in the BS chloroplasts and the total amount of GDC in the BS mitochondria. The variations in the structural and biochemical features involved in photorespiration found among C₄ species might reflect differences in the O₂/CO₂ partial pressure and in the potential photorespiratory capacity of the BS cells.Abbreviations BS Bundle sheath - GDC Glycine decarboxylase - M Mesophyll - NAD-ME NAD-malic enzyme - NADP-ME NADP-malic enzyme - PCK Phosphoenolpyruvate carboxykinase     

Distribution of Plasma Membrane H+-ATPase and Polar Current Patterns in Leaves and Stems of Elodea canadensis*

The proton pumping ATPase in the plasma membrane of Elodea canadensis is believed to play a major role in inorganic carbon acquisition. To investigate potentially different carbon uptake strategies within the same plant, plasma membrane H⁺-ATPase distribution and polar current patterns were investigated in Elodea leaves and stems. Specific activity of plasma membrane H⁺-ATPase in leaf microsomal fractions was tenfold higher than in stem derived microsomes. Probing western blots with a monoclonal antibody specific for plasma membrane H⁺-ATPase, yielded strongly visible double bands at 100 kDa in leaf microsome preparations, whereas little antigen was detected in analogous stem microsome preparations. Using the same plasma membrane H⁺-ATPase specific antibody on tissue sections, the enzyme was found almost exclusively localized at the border of cells at the lower leaf surface. A positive ion current leaving the lower leaf surface was measured, using a vibrating probe device. Part of this current entered the upper leaf surface and part of it the internodes of the stem. The experimental results support the view, that Elodea leaves have different means of inorganic carbon uptake than stem internodes.     

Periplast development in Cryptophyceae II. Development of the inner periplast component inRhinomonas pauca,Proteomonas sulcata [haplomorph],Rhodomonas baltica,andCryptomonas ovata

Summary The inner periplast component (IPC) of numerous cryptomonads is composed of discrete inner plates, situated beneath (and intimately associated with) the plasma membrane (PM). Freeze-fracture images reveal that the PM is organized into a series of ordered structural domains, which directly correspond in size and shape to the underlying inner plates. Freeze-fracture images are used here to compare IPC arrangement inRhinomonas pauca, Proteomonas sulcata [haplomorph],Rhodomonas baltica, andCryptomonas ovata, and to examine development of inner plates in these cryptomonads. In all genera examined, the IPC is highly ordered across most of the cell periphery but appears to be modified adjacent to the vestibulum and mid-ventral line, which represent the anamorphic zones. Variations in the size and shape of PM domains in these regions suggest that development of the IPC occurs within anamorphic zones, by the de novo formation and enlargement of inner plates throughout the cell cycle.     

The surface periplast component of the protistKomma caudata (Cryptophyceae) self-assembles from a secreted high-molecular-mass polypeptide

Summary The cell covering of the cryptomonadKomma caudata (Geitler) Hill is a trilaminar structure consisting of a surface periplast component (SPC) and an inner periplast component (IPC) that sandwich the plasma membrane. In order to investigate the development of the periplast, we have raised monoclonal antibodies against the cell surface ofK. caudata. Immunoblot analyses using one of these antibodies, K1/D.10, showed that it labeled a high-molecular-mass polypeptide. Immunofluorescence and pre- and post-embedding immunogold labeling studies demonstrated that the antibody recognized sites on the cell surface corresponding to the SPC plates and anotherK. caudata cell surface component, the rosulate scales. Labeling was also detected on surface domains devoid of periplast, namely the vestibular/gullet region of the cell. Post-embedding immunocytochemistry revealed that intracellular sites labeled with K1/D.10 included the Golgi apparatus and its associated vesicles. We propose that the subunits of theK. caudata cell covering are antigenically related molecules and that they self-assemble on the cell surface after secretion via the endomembrane system and deployment at the vestibular/gullet region or, in dividing cells, the cytokinetic furrow.     

Structure and immunocytochemical localization of photosynthetic enzymes in the lamina joint and sheath pulvinus of the C4 grass Arundinella hirta

The C₄ grass Arundinella hirta exhibits a unique C₄ anatomy, with isolated Kranz cells (distinctive cells) and C₄-type expression of photosynthetic enzymes in the leaf sheath and stem as well as in the leaf blade. The border zones between these organs are pale green. Those between the leaf blade and sheath and between the sheath and stem are called the lamina joint and sheath pulvinus, respectively, and are involved in gravity sensing. We investigated the structure and localization of C₃ and C₄ photosynthetic enzymes in these tissues. In both zones the epidermis lacked stomata. The inner tissue was composed of parenchyma cells and vascular bundles. The parenchyma cells were densely packed with small intercellular spaces and contained granal chloroplasts with large starch grains. No C₄-type cellular differentiation was recognized. Western blot analysis showed that the lamina joint and pulvinus accumulated substantial amounts of phosphoenolpyruvate carboxylase (PEPC), pyruvate,Pi dikinase (PPDK), and ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco). Immunogold electron microscopy revealed PEPC in the cytosol and both PPDK and rubisco in the chloroplasts of parenchyma cells, suggesting the occurrence of C₃ and C₄ enzymes within a single type of chlorenchyma cell. These data indicate that the lamina joint and pulvinus have unique expression patterns of C₃ and C₄ enzymes, unlike those in C₄-type anatomy.     

CRYPTOGLENA PIGRA: A EUGLENOID WITH ONE CHLOROPLAST12

The taxonomic status of Cryptoglena pigra Ehrb., interpreted from observations based on bright-field microscopy, has been uncertain. Examination with the electron microscope of a clone of C pigra isolated by E. G. Pringsheim reveals certain features which, collectively, are distinctly euglenoid: periplast associated with muciferous bodies and subpellicular microtubules; canal and reservoir with microtubules; one flagellum with a swelling and emergent through a canal, and a second flagellum without a swelling and nonremergent; stigma (eyetpot) closely apprrssed to but not part of the chloroplast; nucleus with permanently condensed chromosomes attached to the inner nuclrar membrane; mitochondria with disc-shaped cristae constricted at the base; chloroplast with thylakoids often in triplets; and paramylon grains in the cytoplasm. Unlike most euglenoids, C. pigra possesses a single chloroplast that in transverse thin sections is U-shaped.     

GRASS LEAF ULTRASTRUCTURAL VARIATIONS

The results of an ultrastructural examination of leaf anatomy and cytology of plastids of both non-Kranz and Kranz grasses are presented. Variations of Kranz anatomy examined include agranal and two granal conditions of kranz-cell chloroplasts, details of the two typical parenchyma sheaths of Aristida, and the “distinctive cells” of species of some small panicoid tribes. These conditions are discussed in relation to C₄ photosynthesis, evolution, and classification.     

The mode of action of fatty alcohols on leaf tissue

The mode of action of a mixture of C₈ and C₁₀ fatty alcohols, formulated in polyoxyethyelene (20) sorbitan mono-oleate (SMO) and used as an emulsion (FAE) to inhibit axillary bud (sucker) growth in tobacco production, was studied using infrared spectroscopy (NIR), photoacoustic spectroscopy (PAS), electrical resistance, and the ability of treated cells to reverse plasmolysis on leaf tissues fromNicotiana tabacum L. and other dicotyledonous species. NIR spectra showed that isolated cuticles were affected optically when treated with FAE, but did not dissolve. PAS absorbances in the UV of isolated cuticles and of epidermal peels were similar and showed that cuticles were homogeneous, unilamellar structures. In intact leaf segments, it was possible, over time using PAS absorbances in the visible region, to separate absorbance of the surface components (cuticle) from the absorption of chlorophyll and other subsurface components and to monitor the penetration by FAE into the leaf. Penetration of the FAE to the subcuticular cells took approximately 2 h. Electrical resistance measurements of FAE-treated isolated midveins of tobacco leaves decreased with time, indicating that the plasma membranes of the cells became leaky. The effect of FAE on plasma membranes of cells was confirmed withElodea sp. where leaf cells after treatment with 1 and 5% FAE lost the ability with time to plasmolyze upon exposure to a 10% solution of Ca(NO₃)₂. The results of the various studies were interpreted to mean that at the labeled concentration (4–5%) for use in the control of axillary bud growth on decapitated tobacco, FAE passed through the cuticle without disrupting it. However, the plasma membranes of the subtending cells were altered so that, in time, bud tissues desiccated (appeared burned) and growth of the sucker was controlled.Cooperative investigations of the Agricultural Research Service of the United States Department of Agriculture with the Agricultural Research Service of the North Carolina State University and the National Institutes of Environmental Health Sciences of the Department of Health and Human Services. The use of trade names in this publication does not imply endorsement by USDA, NCSU, and NIEHS of products named nor criticism of similar ones not mentioned.     

Protein kinase C enhances plasma membrane expression of cardiac L-type calcium channel,CaV1.2

L-type-voltage-dependent Ca²⁺ channels (L-VDCCs; Ca_V1.2, α_1C), crucial in cardiovascular physiology and pathology, are modulated via activation of G-protein-coupled receptors and subsequently protein kinase C (PKC). Despite extensive study, key aspects of the mechanisms leading to PKC-induced Ca²⁺ current increase are unresolved. A notable residue, Ser1928, located in the distal C-terminus (dCT) of α_1C was shown to be phosphorylated by PKC. Ca_V1.2 undergoes posttranslational modifications yielding full-length and proteolytically cleaved CT-truncated forms. We have previously shown that, in Xenopus oocytes, activation of PKC enhances α_1C macroscopic currents. This increase depended on the isoform of α_1C expressed. Only isoforms containing the cardiac, long N-terminus (L-NT), were upregulated by PKC. Ser1928 was also crucial for the full effect of PKC. Here we report that, in Xenopus oocytes, following PKC activation the amount of α_1C protein expressed in the plasma membrane (PM) increases within minutes. The increase in PM content is greater with full-length α_1C than in dCT-truncated α_1C, and requires Ser1928. The same was observed in HL-1 cells, a mouse atrium cell line natively expressing cardiac α_1C, which undergoes the proteolytic cleavage of the dCT, thus providing a native setting for exploring the effects of PKC in cardiomyocytes. Interestingly, activation of PKC preferentially increased the PM levels of full-length, L-NT α_1C. Our findings suggest that part of PKC regulation of Ca_V1.2 in the heart involves changes in channel's cellular fate. The mechanism of this PKC regulation appears to involve the C-terminus of α_1C, possibly corroborating the previously proposed role of NT-CT interactions within α_1C.     

MICROMORPHOLOGY OF THE PERIPLAST OF CHROOMONAS SP. (CRYPTOPHYCEAE)1,2

An ultrastructural examination of the periplast of Chroomonas sp. revealed a surface pattern composed of rows of plate areas. The plate areas are delineated by a series of ridges, which emanate from a common line at the posterior cell end, and lateral grooves which intersect the anterior-posterior ridges. Small ejectosomes (trichocysts) are generally located at the intersection of the lateral grooves and the ridges. Size of the plate areas varies, being smallest at the posterior and anterior ends and largest in the midregion of the cell. The average plate area is 1 μ in length and 0.7 μ in width. In section the periplast is seen to consist of 3 intimately attached layers of which the middle (plasma membrane) layer is continuous with the gullet region, flagella, and ejectosome chambers. Trypsin digestion resulted in the disappearance of the inner and outer layers, and in the loss of periplast stiffness.     

Periplaststruktur und Organisation der Plasmamembran vonRhodomonas spec. (Cryptophyceae)

Summary The periplast of the CryptophyceaeRhodomonas has a hexagonal substructure. This substructure is caused by periplast plates. In freeze fracture replicas of the plasma membrane, there are corresponding hexagonal areas with numerous particles. These areas are separated by regions with less particles. Aggregates of particles, partly rosette-like, indicate insertion sites of ejectisomes.

     

Periplast development in cryptophyceae I. Changes in periplast arrangement throughout the cell cycle

Summary The cell covering (or periplast) of many cryptomonads consists of discrete plate areas precisely arranged over most of the cell periphery. Developmental changes in periplast arrangement that occur throughout the cell cycle are examined here forKomma caudata andProteomonas sulcata [haplomorph]. In both cryptomonads, pole reversal occurs during cytokinesis, necessitating major realignment of the plate areas. Growth of the periplast occurs by addition of new plate areas to specialized regions (termed anamorphic zones) located around the vestibular margins and along the mid-ventral line of cells. Development of the periplast from these regions enables elongation and lateral expansion of cryptomonads throughout cell growth. Observed differences in cell division and periplast development between these genera are closely associated with variations in the arrangement of anamorphic zones.     

The effect of light on membrane potential, apoplastic pH and cell expansion in leaves of Pisum sativum L. var. Argenteum.

The connection between three light responses of green leaf cells-membrane potential (V_m), H⁺ net efflux and growth, was analyzed. Illumination of mesophyll cells in leaves from Argenteum peas caused two rapid responses: (i) a de- and repolarization of V_m and (ii) an alkalinization of the apoplast. The rapid responses were completely eliminated by the photosynthetic inhibitor 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU) but not affected by ortho-vanadate, an inhibitor of the plasma membrane (PM) H⁺-ATPase. The rapid changes were followed by a set of delayed responses: (i) a slow, gradual hyperpolarization of V_m, (ii) a gradual acidification of the mesophyll apoplast and (iii) an increased rate of elongation. These three light responses persisted under DCMU but were completely eliminated by vanadate. The data show that the delayed (in contrast to the rapid) responses were due to a stimulation of PM H⁺ pumps which occurred independently of non-cyclic photosynthetic electron transport and the “dark” processes depending on it. When the rapid responses were blocked by DCMU, light-induced acidification, hyperpolarization of the membrane potential and growth proceeded simultaneously. A shared (4-min) lag phase indicated slower signal processing in mesophyll than in epidermal cells where light stimulation of PM H⁺ pumps was rapid. Received: 3 September 1998 / Accepted: 15 October 1998     

pH-Induced Proton Permeability Changes of Plasma Membrane Vesicles

In vivo studies with leaf cells of aquatic plant species such as Elodea nuttallii revealed the proton permeability and conductance of the plasma membrane to be strongly pH dependent. The question was posed if similar pH dependent permeability changes also occur in isolated plasma membrane vesicles. Here we report the use of acridine orange to quantify passive proton fluxes. Right-side out vesicles were exposed to pH jumps. From the decay of the applied ΔpH the proton fluxes and proton permeability coefficients (P_H+) were calculated. As in the intact Elodea plasma membrane, the proton permeability of the vesicle membrane is pH sensitive, an effect of internal pH as well as external pH on P_H+ was observed. Under near symmetric conditions, i.e., zero electrical potential and zero ΔpH, P_H+ increased from 65 × 10⁻⁸ at pH 8.5 to 10⁻¹ m/sec at pH 11 and the conductance from 13 × 10⁻⁶ to 30 × 10⁻⁴ S/m². At a constant pH _i of 8 and a pH _o going from 8.5 to 11, P_H+ increased more than tenfold from 2 to 26 × 10⁻⁶ m/sec. The calculated values of P_H+ were several orders of magnitude lower than those obtained from studies on intact leaves. Apparently, in plasma membrane purified vesicles the transport system responsible for the observed high proton permeability in vivo is either (partly) inactive or lost during the procedure of vesicle preparation. The residue proton permeability is in agreement with values found for liposome or planar lipid bilayer membranes, suggesting that it reflects an intrinsic permeability of the phospholipid bilayer to protons. Possible implications of these findings for transport studies on similar vesicle systems are discussed. Received: 5 April 1995/Revised: 28 March 1996     

The mode of action of fatty alcohols on leaf tissue

The mode of action of a mixture of C₈ and C₁₀ fatty alcohols, formulated in polyoxyethyelene (20) sorbitan mono-oleate (SMO) and used as an emulsion (FAE) to inhibit axillary bud (sucker) growth in tobacco production, was studied using infrared spectroscopy (NIR), photoacoustic spectroscopy (PAS), electrical resistance, and the ability of treated cells to reverse plasmolysis on leaf tissues fromNicotiana tabacum L. and other dicotyledonous species. NIR spectra showed that isolated cuticles were affected optically when treated with FAE, but did not dissolve. PAS absorbances in the UV of isolated cuticles and of epidermal peels were similar and showed that cuticles were homogeneous, unilamellar structures. In intact leaf segments, it was possible, over time using PAS absorbances in the visible region, to separate absorbance of the surface components (cuticle) from the absorption of chlorophyll and other subsurface components and to monitor the penetration by FAE into the leaf. Penetration of the FAE to the subcuticular cells took approximately 2 h. Electrical resistance measurements of FAE-treated isolated midveins of tobacco leaves decreased with time, indicating that the plasma membranes of the cells became leaky. The effect of FAE on plasma membranes of cells was confirmed withElodea sp. where leaf cells after treatment with 1 and 5% FAE lost the ability with time to plasmolyze upon exposure to a 10% solution of Ca(NO₃)₂. The results of the various studies were interpreted to mean that at the labeled concentration (4–5%) for use in the control of axillary bud growth on decapitated tobacco, FAE passed through the cuticle without disrupting it. However, the plasma membranes of the subtending cells were altered so that, in time, bud tissues desiccated (appeared burned) and growth of the sucker was controlled.     

Evidence for the expression of morphological and biochemical characteristics of C3-photosynthesis in chlorohyllous callus cultures of Zea mays

A Zea mays callus culture containing chlorophyll was established and grown photomixotrophically. Cell chloroplast structure, and pigment and soluble protein contents were examined. Expression of some key enzymes of C₄ carbon metabolism was compared with that of etiolated (heterotrophic) and green photoautotrophic leaves. Chlorophyll content of the callus was 15–20% that of green leaves. Soluble protein content of callus was half that of leaf cells. Electron microscopic observations showed that green callus cells contained only typical granal chloroplasts. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.38) activities in green callus were ca 30% those of green leaves but 2–3 times higher than in etiolated leaves. Quantitative enzyme protein determination, using antibodies specific to maize leaf Rubisco showed that the chloroplastic carboxylase represented about 7% of total soluble protein in green callus, in parallel to its low chlorophyll content. The specific activity of Rubisco in callus and leaves was unchanged. Phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) activity in green callus was about 20% that of green leaves and similar to that measured in etiolated leaves. Apparent K_m (PEP) values (0.08 mM) for PEPC isolated from green callus and etiolated leaves were very different from values (0.5 mM) obtained with PEPC from green leaves. These kinetic characteristics together with the absence of inhibition by malate and activation by glucose-6-phosphate suggest that the properties of PEPC isolated from green callus and etiolated maize leaves are very similar to those of PEPPC from C₃ plants. Using PEPC antibodies specific to green maize leaf enzyme, immunotitration of PEPC preparations containing identical enzyme units allowed complete precipitation of the green leaf enzyme with increasing antibody volumes. In contrast, 60–70% of the activity of PEPC from etiolated and green callus was inhibited, suggesting low affinity for the maize green leaf PEPC antiserum (typical C₄ form). Ouchterlony double diffusion tests revealed only partial recognition of PEPC in green callus and etiolated leaves. NAD-malate dehydrogenase (NAD-MDH, EC 1.1.1.37) activity in callus was 2 and 3 times higher, respectively, than in etiolated and green leaves. NADP-malic enzyme (NADP-ME, EC 1.1.1.40) activity in callus cultures was much lower than in green leaves. All our data support the hypothesis that cultures of fully dedifferentiated chlorophyllous tissues of Zea mays possess a C₃-like metabolism.